Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. PTB is one of the major regulators of alternate splicing. However our understanding of how PTB and many other RNA binding proteins achieve specificity is incomplete. The goal of this research proposal is to develop efficient methods to identify the sequence elements that govern splicing in the genome and to understand how these elements work together. While this method is broadly applicable, this proposal focuses exclusively on PTB binding pre-mRNA by building on preliminary data obtained from a pilot study. There are two complementary parts to this project. The first aim describes a means for the experimental validation of thousands of candidate ligands for RNA binding proteins. The binding of factors to these ligands are tested in a novel massively parallel high-throughput binding assay. The next phase is to incorporate the PTB binding features that we learn from the screen into a probabilistic model. We then test the model in vivo by CLIP and functional assays. PUBLIC HEALTH RELEVANCE: The vast majority of human genes undergo extensive processing before they are expressed as proteins. Most genes are alternatively processed across different tissues. PTB is the dominant splicing repressor that regulates alternative splicing events. This project seeks to define the sequence, structural and higher order architectures of PTB binding sites in human cells.